CN113106048B

CN113106048B - Radiation-resistant genetically engineered bacterium Deino-dsrA as well as construction method and application thereof

Info

Publication number: CN113106048B
Application number: CN202110392388.4A
Authority: CN
Inventors: 肖方竹; 何淑雅; 彭国文; 朱琦琦; 罗佳琦; 刘军
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2021-04-13
Filing date: 2021-04-13
Publication date: 2022-09-20
Anticipated expiration: 2041-04-13
Also published as: CN113106048A

Abstract

The invention provides a radiation-resistant genetically engineered bacterium Deino-dsrA and a construction method and application thereof. The construction method comprises the following steps: using the genome DNA containing the reducing gene dsrA as an amplification template to carry out PCR amplification, and purifying and recovering a PCR product to obtain the target gene dsrA; carrying out double digestion on target genes dsrA and pRADK plasmids by endonuclease Nde I and endonuclease BamH I, purifying and recovering, and connecting by ligase to obtain a recombinant vector pRADK-dsrA; and transforming the recombinant vector pRADK-dsrA into radiation-resistant deinococcus sp to construct a radiation-resistant genetically engineered bacterium Deino-dsrA, performing PCR identification, and screening out a strain with positive gene amplification. The radiation-resistant genetically engineered bacterium Deino-dsrA constructed by the invention is applied to the field of low-concentration uranium polluted water body remediation, the enrichment rate of uranium can reach more than 90%, and the radiation-resistant genetically engineered bacterium Deino-dsrA has the advantages of good enrichment effect, low cost, environmental friendliness and the like.

Description

Radiation-resistant genetically engineered bacterium Deino-dsrA as well as construction method and application thereof

Technical Field

The invention belongs to the technical field of microbial remediation treatment, and particularly relates to a radiation-resistant genetically engineered bacterium Deino-dsrA, a construction method and application thereof in uranium-polluted water remediation.

Background

Uranium is a naturally radioactive element widely distributed in rocks and soil. In recent years, the nuclear industry has been rapidly developed, and the demand of uranium as an important nuclear industry raw material has been increasing, and at the same time, water pollution caused by uranium has been receiving more and more attention. The sources of uranium in the water body mainly comprise uranium ore mining, smelting and processing, uranium leakage caused by abnormal accidents of nuclear power stations, production and test of nuclear weapons, use of depleted uranium weapons and the like. Uranium has both chemical toxicity and radiotoxicity hazards, and can enter the body through respiratory tracts and digestive tracts, and easily soluble uranium compounds can enter the body through intact skin, so that internal irradiation and chemical toxicity are generated, damages can be generated to the kidney, immune system, nervous system and fertility of people, and gene mutation and cancers can be caused. At present, the method for purifying uranium in uranium-containing wastewater mainly comprises the following steps: chemical precipitation, adsorption, ion exchange, solvent extraction, biological treatment, etc. Due to the low economics and effectiveness of physicochemical treatment methods, microbial treatment is a preferred method due to its low cost, high efficiency, ease of culture, and the like.

Deinococcus Radiodurans (DR) is a coccus or nonsporium bacilli, is nonpathogenic, has extremely strong radiation, oxidation and DNA damage repair resistance, and is considered to be the most radiation-resistant microorganism on earth. Deinococcus radiodurans has strong resistance to dryness, ionizing radiation, oxygen free Radicals (ROS), toxic chemical substances and the like, and is applied to removing heavy metal ions such as cobalt, iron and the like in radioactive nuclear polluted wastewater at present. The wild deinococcus radiodurans has a certain enrichment effect on uranium, but still has the defect of low enrichment rate, and cannot meet the requirement of large-scale industrial application.

Disclosure of Invention

The invention aims to provide a radiation-resistant genetically engineered bacterium Deino-dsrA constructed in a transgenic mode, a construction method and application thereof in uranium-polluted water body remediation.

In order to achieve the aim, the invention provides a construction method of a radiation-resistant genetically engineered bacterium Deino-dsrA, which comprises the following steps:

(1) using the genome DNA containing the reducing gene dsrA as an amplification template to carry out PCR amplification, and purifying and recovering a PCR product to obtain the target gene dsrA;

(2) carrying out double digestion on target genes dsrA and pRADK plasmids by endonuclease Nde I and endonuclease BamH I, purifying and recovering, and connecting by ligase to obtain a recombinant vector pRADK-dsrA;

(3) and transforming the recombinant vector pRADK-dsrA into deinococcus radiodurans to construct a radioresistant genetic engineering bacterium Deino-dsrA, carrying out PCR identification, and screening out a strain with positive gene amplification.

In a specific embodiment, in step (1), the nucleotide sequences of the primers amplified by PCR are as follows:

the sequence of the upstream primer dsrA-F is as follows:

5’-CCCTGCAGGTCGAATCGGATCCCCAAGGCAGGCTTCAG-3’，

the downstream primer dsrA-R sequence is as follows:

5’-CTCACAGGAGGACCCCATATGCATGTGGAGGTAGGCA-3’；

wherein, the underlined base part in the upstream primer dsrA-F sequence is a BamHI restriction site, and the underlined base part in the downstream primer dsrA-R sequence is an NdeI restriction site.

In a specific embodiment, said genomic DNA containing the reducing gene dsrA is extracted from sulfate-reducing bacteria.

The invention also provides a radiation-resistant genetically engineered bacterium Deino-dsrA constructed by the construction method.

The invention also provides application of the radiation-resistant genetically engineered bacterium Deino-dsrA in uranium polluted water body remediation.

The invention also provides a preparation method of the uranium-polluted water body repairing agent, which comprises the steps of culturing the radiation-resistant genetically engineered bacterium Deino-dsrA in a TGY solid culture medium for resuscitation, selecting a monoclonal strain to inoculate the strain in a TGY liquid culture medium, and carrying out expanded culture until the OD (OD) of the strain liquid is obtained ₆₀₀ The value is 0.9 to 1.0; then diluting the bacterial liquid to obtain OD ₆₀₀ The value is 0.5-0.6, and the bacterial suspension is a uranium-polluted water body repairing agent.

In a specific embodiment, the pH value of the TGY solid medium is 6.2-6.6, and the TGY solid medium comprises the following components in percentage by mass: 0.5% of trypsin jelly, 0.3% of yeast extract, 0.1% of D-glucose, 1.5% of agar and the balance of ultrapure water.

In a specific embodiment, the pH value of the TGY liquid culture medium is 6.2-6.6, and the TGY liquid culture medium comprises the following components in percentage by mass: 0.5% of trypsin jelly, 0.3% of yeast extract, 0.1% of D-glucose and the balance of ultrapure water.

The invention also provides a method for repairing the uranium-polluted water body, which comprises the steps of mixing the bacterial suspension prepared by the preparation method and a uranium solution according to a preset proportion, adjusting the pH value to be 4.8-6, and then placing the mixture in a constant-temperature air bath shaking table at 29-31 ℃ for enrichment reaction, wherein the time of the enrichment reaction is 55-105 min.

In a specific embodiment, the initial mass concentration of uranium in the uranium solution is 10-30 mg/L, and the preset ratio is 2: 1, the rotating speed of the constant-temperature air bath shaking table is 220 r/min.

The beneficial effects of the invention at least comprise:

1. the invention takes genome DNA containing reducing gene dsrA as an amplification template, PCR amplification is carried out, a PCR product is purified and recovered to obtain a target gene dsrA, then the target gene dsrA is connected with a pRADK carrier to obtain a recombinant carrier pRADK-dsrA, and the recombinant carrier pRADK-dsrA is transferred into deinococcus radiodurans, so as to construct radioresistant genetically engineered bacterium Deino-dsrA containing the reducing gene dsrA; the radiation-resistant genetically engineered bacterium Deino-dsrA is applied to the field of low-concentration uranium polluted water body remediation, the enrichment rate of uranium can reach over 90 percent and is far greater than the enrichment rate of 72.02 percent of wild type radiation-resistant deinococcus, and the method has the advantages of good enrichment effect, low cost, environmental friendliness and the like.

2. Culturing radiation-resistant genetically engineered bacterium Deino-dsrA in TGY solid culture medium for resuscitation, selecting monoclonal strain, inoculating in TGY liquid culture medium, and performing amplification culture to obtain bacterial liquid OD ₆₀₀ The value is 0.9 to 1.0; then diluting the bacterial liquid to obtain OD ₆₀₀ The value is 0.5 ~ 0.6 bacterial suspension, bacterial suspension takes place enrichment reaction with uranium solution as uranium contaminated water body remediation agent, because it is the suspension state, can with uranium solutionThe uranium solution is fully contacted, thereby improving the efficiency and enrichment rate of the enrichment reaction.

Drawings

FIG. 1 is a diagram showing the effect of uranium enrichment of a uranium-polluted water restoration agent provided by the invention at different pH values;

fig. 2 is a diagram of the effect of uranium enrichment of the uranium polluted water restoration agent provided by the invention under the condition of uranium solutions with different initial concentrations;

FIG. 3 is a diagram showing the effect of uranium enrichment in the uranium-polluted water restoration agent provided by the invention in different enrichment reaction times;

FIG. 4 is a scanning electron microscope image before and after uranium enrichment by the radiation-resistant genetically engineered bacterium Deino-dsrA constructed in the embodiment of the present invention.

Detailed Description

Example 1:

construction of radiation-resistant genetically engineered bacterium Deino-dsrA

Main microbial agents (reagents): deinococcus Radiodurans (DR) (preservation number: CGMCC1.633), Sulfate Reducing Bacteria (SRB) and prokaryotic expression vector pRADK plasmids are all subcultured in nuclear radiation biological effect key laboratory of southern China university; the bacterial DNA extraction kit is purchased from Beijing Tiangen biology, Inc., the PCR amplification primer is synthesized by Jinwei Zhi, the Nde I enzyme, the BamH I enzyme and the T4DNA ligase are purchased from Biyunnan biology, Inc., the high-purity plasmid small-extraction kit is purchased from Beijing kang, Inc., and other reagents are domestic analytical purifications.

The construction method of the radiation-resistant genetically engineered bacterium Deino-dsrA comprises the following steps:

extracting genomic DNA containing key reducing gene dissimilatory sulfite reductase A (dsrA) from sulfate reducing bacteria as a template of PCR amplification reaction, and then designing and synthesizing PCR amplification primers, wherein:

the sequence of the upstream primer dsrA-F is as follows:

5’-CCCTGCAGGTCGAATCGGATCCCCAAGGCAGGCTTCAG-3’(SEQ ID NO.1)；

the downstream primer dsrA-R sequence is as follows:

5’-CTCACAGGAGGACCCCATATGCATGTGGAGGTAGGCA-3'(SEQ ID NO.2)；

the underlined base part in the upstream primer dsrA-F sequence is a BamHI restriction site, and the underlined base part in the downstream primer dsrA-R sequence is an Nde I restriction site.

Adding the PCR amplification primer to perform PCR amplification reaction by taking the genomic DNA as a template, purifying and recovering a PCR product to obtain a target gene dsrA, performing double enzyme digestion on the pRADK plasmid and the target gene dsrA by using endonuclease Nde I and BamH I, and performing gel electrophoresis and gel recovery on the double enzyme digestion product; and connecting the recovered enzyme-digested target gene dsrA with a vector plasmid pRADK by using T4DNA ligase to obtain a recombinant vector pRADK-dsrA, transforming the recombinant vector pRADK-dsrA into radiation-resistant deinococcus sp to construct a radiation-resistant gene engineering bacterium Deino-dsrA, performing PCR identification, and then sending the plasmid of the strain with the gene amplified to be positive to Shanghai's company for sequencing identification, wherein the result shows that the gene insertion position is correct, the frameshift mutation does not occur, and the vector construction is successful.

Example 2

Preparation of uranium polluted water restoration agent by using radiation-resistant genetically engineered bacterium Deino-dsrA constructed in example 1

Culturing radiation-resistant genetically engineered bacterium Deino-dsrA in TGY solid culture medium for resuscitation, selecting monoclonal strain, inoculating in TGY liquid culture medium, and performing amplification culture to obtain bacterial liquid OD ₆₀₀ The value was 1, which was then made to OD with 0.9% physiological saline ₆₀₀ The value is 0.5, and the bacterial suspension is a uranium-polluted water body repairing agent. Wherein the pH value of the TGY solid culture medium is 6.4, and the TGY solid culture medium comprises the following components in percentage by mass: 0.5% of Tryptone (Tryptone), 0.3% of Yeast extract (Yeast extract), 0.1% of D-glucose (D-glucose), 1.5% of Agar (Agar) and the balance of ultrapure water; the pH value of the TGY liquid culture medium is 6.4, and the TGY liquid culture medium comprises the following components in percentage by mass: 0.5% of trypsin jelly, 0.3% of yeast extract, 0.1% of D-glucose and the balance of ultrapure water.

Examples 3 to 21

Uranium in uranium solution is enriched by using the uranium-polluted water restoration agent prepared in example 2

The experimental method comprises the following steps:

mixing the bacterial suspension prepared in the embodiment 2 and a uranium solution according to a preset proportion, adjusting the pH value, and then placing the mixture in a 30 ℃ constant temperature air bath shaking table for enrichment reaction, wherein the rotating speed of the constant temperature air bath shaking table is set to be 220 r/min; after the enrichment reaction is carried out for a preset time, centrifuging to obtain 1mL of supernatant, measuring the absorbance of the supernatant by adopting an ultraviolet-visible spectrophotometry, calculating to obtain the concentration of a uranium solution in the supernatant according to the absorbance of the supernatant, and finally calculating to obtain the enrichment ratio P according to a formula (1), wherein the formula (1) is as follows:

in the formula: c ₀ Is the initial mass concentration of uranium, mg/L; c _t The residual mass concentration of uranium in the solution at the time t is mg/L; p is the enrichment ratio.

Example 3

Taking 10mL of uranium solution with pH5.0 and 20mL of OD ₆₀₀ Adding the bacterial suspension with the value of 0.5 into a 100mL conical flask with a stopper to ensure that the initial mass concentration of uranium in the uranium solution is 30 mg/L; then placing the conical bottle with the plug in a 30 ℃ constant temperature air bath shaking table, and shaking for 60min at a constant temperature of 220 r/min; centrifuging to obtain 1mL of supernatant, adding the supernatant into a colorimetric tube with a plug, adding 1mL of acetic acid-sodium acetate buffer solution with the mass fraction of 0.05 percent of azoarsine III and the pH value of 4, fixing the volume to 10mL, reversing the solution from top to bottom, uniformly mixing, standing for 10min, measuring the absorbance of the solution at the wavelength of 652nm by using an ultraviolet-visible spectrophotometer, and calculating the enrichment rate P to be 92.45 percent based on the absorbance.

Examples 4 to 9 experiments were carried out in the same manner as in example 3, except that the pH of the uranium solution added was different, and the pH of the uranium solution added in example 3 was 5.0; example 4 the pH of the uranium solution added was 2.0; example 5 the pH of the added uranium solution was 3.0; example 6 the pH of the added uranium solution was 4.0; example 7 the pH of the added uranium solution was 6.0; example 8 the pH of the added uranium solution was 7.0; example 9 the pH of the added uranium solution was 8.0; the influence of pH on the enrichment effect is shown in detail in fig. 1, which is drawn by using the pH of the uranium solutions of examples 3 to 9 as the horizontal and vertical scales and the corresponding enrichment ratio P as the vertical coordinate.

As can be seen from FIG. 1, the enrichment ratio can reach 90% or more at a pH of 4.8 to 6. In the environment with too low pH value, U (VI) is dissolved in uranium solution as UO ₂ ²⁺ In the form of (1) with a large amount of H in solution ⁺ Occupy nucleophilic sites on the surface of the engineering bacteria agent to generate mutual competition, so that UO is generated ₂ ²⁺ Can not be well contacted with the nucleophilic sites on the surface of the engineering bacteria agent. In an environment with an excessively high pH value, hydrolysis of uranyl ions in the solution generates uranyl hydrate positive ions with a large space structure, including [ UO ] ₂ OH] ⁺ ，[(UO ₂ ) ² (OH) ₂ ] ²⁺ ，[(UO ₂ ) ³ (OH) ₅ ] ⁺ ，[(UO ₂ ) ₄ (OH) ₇ ] ⁺ Etc., occupy more active sites on the cell wall surface, resulting in a decreased enrichment rate. Therefore, the pH value of the uranium solution is preferably 4.8-6.

Examples 10 to 15 experiments were carried out in the same manner as in example 3, except that the initial uranium mass concentration was different, and that in example 3 the initial uranium mass concentration was 30 mg/L; example 10 the initial mass concentration of uranium is 10 mg/L; example 11 the initial mass concentration of uranium is 20 mg/L; example 12 the initial mass concentration of uranium is 40 mg/L; example 13 the initial mass concentration of uranium is 50 mg/L; example 14 the initial mass concentration of uranium is 60 mg/L; example 15 the initial mass concentration of uranium is 70 mg/L; the graph of the influence of the initial mass concentration of uranium on the enrichment effect is drawn by taking the initial mass concentration of uranium in example 3 and examples 10 to 15 as a horizontal ordinate and taking the corresponding enrichment ratio P value as a vertical ordinate, and is shown in fig. 2 in detail.

As can be seen from fig. 2, the enrichment rate shows a decreasing trend as the initial mass concentration of uranium in the uranium solution increases. When the addition amount of the bacteria is certain and the initial mass concentration of uranium in the uranium solution in the solution is low, cell wall surface active sites are sufficient, and uranyl ions are fully contacted with the bacteria, so that the enrichment rate is high. Increasing the initial mass concentration of uranium in the uranium solution, gradually saturating enrichment sites on the surfaces of the thalli, and enabling uranyl ions to occupy limited active sites to balance enrichment. Therefore, the initial mass concentration of the uranium in the uranium solution is preferably 10-30 mg/L.

Examples 16 to 21 experiments were carried out in the same manner as in example 3 except that the preset time for the enrichment reaction was different, and the time for the enrichment reaction in example 3 was 30 min; example 16 enrichment reaction time 15 min; example 17 enrichment reaction time 45 min; example 18 enrichment reaction time 60 min; example 19 enrichment reaction time 75 min; example 20 enrichment reaction time 90 min; example 21 enrichment reaction time 105 min; the graph of the effect of the enrichment reaction time on the enrichment effect, which is plotted with the enrichment reaction time of example 3 and example 16 to example 21 as the horizontal and vertical scales and the corresponding enrichment ratio P as the vertical coordinate, is shown in detail in fig. 3.

Control group 1 to control group 7

Control group 1 was subjected to the experiment in the same manner as in example 3 except that the radiation-resistant genetically engineered bacterium Deino-dsrA was replaced with a wild-type deinococcus radiodurans.

Experiments are carried out on the control group 2 to the control group 7 according to the same method as the control group 1, and the difference is that the preset time of the enrichment reaction is different, and the enrichment reaction time of the control group 1 is 30 min; the enrichment reaction time of the control group 2 is 15 min; the enrichment reaction time of the control group 3 is 45 min; the enrichment reaction time of the control group 4 is 60 min; the enrichment reaction time of the control group 5 is 75 min; the enrichment reaction time of the control group 6 is 90 min; the enrichment reaction time of the control group 7 is 105 min; the graph showing the effect of the enrichment reaction time on the enrichment effect is shown in detail in FIG. 3, with the enrichment reaction time of the control groups 1 to 7 as the horizontal and vertical scales and the corresponding enrichment ratio P as the vertical coordinate.

As can be seen from FIG. 3, the enrichment ratio of the Control group (Control) is obviously lower than that of the experimental group (Deino-dsrA), and because the radiation-resistant genetically engineered bacterium Deino-dsrA in the experimental group contains dsrA reducing genes, the expressed reductase can reduce U (VI) in the solution into U (IV), so that the U (VI) is more easily precipitated on the surface of the bacteria. And when the enrichment reaction time is 55-105 min, the enrichment rate is more than 90%, and the enrichment reaction time is more preferably 60min in consideration of time cost.

Referring to fig. 4, fig. 4 is a scanning electron microscope image before and after uranium enrichment by radiation-resistant genetically engineered bacteria Deino-dsrA constructed according to an embodiment of the present invention, where fig. 4(a) is a scanning electron microscope image before uranium enrichment by radiation-resistant genetically engineered bacteria Deino-dsrA, and at this time, the surface shape of the bacteria is irregular, so as to provide a larger contact surface area and enrichment sites for uranium enrichment; fig. 4(b) is a scanning electron microscope image of the radiation-resistant genetically engineered bacterium Deino-dsrA corresponding to example 3 after enrichment of uranium, and it can be seen from the image that a large amount of particles appear on the surface of the bacterium body, which indicates that the radiation-resistant genetically engineered bacterium Deino-dsrA is enriched with a certain amount of uranium.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions and substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Sequence listing

<110> university of southern China

<120> radiation-resistant genetically engineered bacterium Deino-dsrA, and construction method and application thereof

<130> 2021

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccctgcaggt cgaatcggat ccccaaggca ggcttcag 38

<210> 2

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctcacaggag gaccccatat gcatgtggag gtaggca 37

Claims

1. A method for constructing a radiation-resistant genetically engineered bacterium Deino-dsrA is characterized by comprising the following steps:

(3) transforming the recombinant vector pRADK-dsrA into deinococcus radiodurans to construct a radioresistant genetic engineering bacterium Deino-dsrA, carrying out PCR identification, and screening out a strain with positive gene amplification;

in the step (1), the nucleotide sequences of the primers amplified by the PCR are as follows:

the sequence of the upstream primer dsrA-F is as follows:

5’-CCCTGCAGGTCGAATCGGATCCCCAAGGCAGGCTTCAG-3’，

the downstream primer dsrA-R sequence is as follows:

5’-CTCACAGGAGGACCCCATATGCATGTGGAGGTAGGCA-3’；

2. The method for constructing the radiation-resistant genetically engineered bacterium Deino-dsrA according to claim 1, wherein the genomic DNA containing a reducing gene dsrA is extracted from sulfate-reducing bacteria.

3. A radiation-resistant genetically engineered bacterium Deino-dsrA, which is constructed by the construction method of any one of claims 1 to 2.

4. The application of the radiation-resistant genetically engineered bacterium Deino-dsrA as claimed in claim 3 in uranium-polluted water body remediation.

5. A preparation method of a uranium polluted water body repairing agent is characterized in that after the radiation-resistant genetically engineered bacterium Deino-dsrA of claim 4 is cultured and recovered in a TGY solid culture medium, a monoclonal strain is selected and inoculated in a TGY liquid culture medium, and the strain is expanded and cultured until the OD (oxygen-dependent density) of a bacterium liquid is obtained ₆₀₀ The value is 0.9 to 1.0; then diluting the bacterial liquid to obtain OD ₆₀₀ The value is 0.5-0.6, and the bacterial suspension is a uranium-polluted water body repairing agent.

6. The preparation method according to claim 5, wherein the TGY solid medium has a pH value of 6.2-6.6, and comprises the following components by mass percent: 0.5% of trypsin jelly, 0.3% of yeast extract, 0.1% of D-glucose, 1.5% of agar and the balance of ultrapure water.

7. The preparation method according to claim 6, wherein the pH value of the TGY liquid medium is 6.2-6.6, and the TGY liquid medium comprises the following components in percentage by mass: 0.5% of trypsin jelly, 0.3% of yeast extract, 0.1% of D-glucose and the balance of ultrapure water.

8. A uranium polluted water body remediation method is characterized in that the bacterial suspension prepared by the preparation method of any one of claims 5 to 7 and a uranium solution are mixed according to a preset proportion, the pH value is adjusted to 4.8-6, and then the mixture is placed in a constant-temperature air bath shaking table at 29-31 ℃ for enrichment reaction, wherein the time of the enrichment reaction is 55-105 min.

9. The method for repairing the uranium-polluted water body according to claim 8, wherein the initial mass concentration of uranium in the uranium solution is 10-30 mg/L, and the preset ratio is 2: 1, the rotating speed of the constant-temperature air bath shaking table is 220 r/min.